Journal of Applied Pharmaceutical Science Vol. 6 (01), pp.

007-014, January, 2016

Available online at http://www.japsonline.com
DOI: 10.7324/JAPS.2016.600102
ISSN 2231-3354

Spectrophotometric Determination of Gentamicin Loaded PLGA

Microparticles and Method Validation via Ninhydrin-Gentamicin
Complex as a Rapid Quantification Approach

Ahmad Fahmi Harun Ismail1, Farahidah Mohamed1*, Luqman Muizzuddin Mohd Rosli1, Mohd Affendi Mohd Shafri2,
Muhammad Salahuddin Haris1, Anugerah Budipratama Adina2
1
Kulliyyah of Pharmacy (KOP), International Islamic University Malaysia (IIUM), Jalan Sultan Ahmad Shah, Bandar Indera Mahkota, 25200 Kuantan,
Pahang Darul Makmur, Malaysia. 2Kulliyyah of Allied Health Sciences (KAHS), International Islamic University Malaysia (IIUM), Jalan Sultan Ahmad
Shah, Bandar Indera Mahkota, 25200 Kuantan, Pahang Darul Makmur, Malaysia.

ARTICLE INFO ABSTRACT

Article history:
Received on: 05/11/2015
Revised on: 09/12/2015
Accepted on: 22/12/2015
Available online: 26/01/2016
Key words:
Spectrophotometry;
Gentamicin; Ninhydrin;
Microparticles; poly(lactic-
co-glycolic acid).
The purpose of this study was to provide a quantification method with rapid, sensitive, reproducible and cost
effective for gentamicin in the form of ninhydrin-gentamicin complex. The utilization of spectrophotometric
module to validate the method development for gentamicin loaded microparticles was intended to provide
alternative quantification method without undermining the sensitivity of the developed method. The
microparticles fabrication process was proven to be suitable in encapsulating gentamicin by using poly(lactic-co-
glycolic acid) PLGA without compromising the efficacy of the antibiotic itself. The linearity of 6 known
concentrations of ninhydrin-gentamicin complex was obtained with the R2 of 0.9998. The interday and intraday
precisions were determined with the acceptance %RSD values of less than 2%. The highest %RSD value was
1.09% which suggested the method to be acceptably precise. The limit of detection (LOD) and limit of
quantification (LOQ) values were recorded to be at 0.016 and 0.196 mg/mL respectively. The % recovery of 4
known concentrations indicated the accuracy of the method was high with the recovery range between 98.66%
and 101.8%. The parameters analyzed in this study were in accordance with ICH Q2 (R1) guidelines. This
quantification method was a promising approach to provide a rapid and cost effective in evaluating gentamicin
concentration for in-vitro applications.

INTRODUCTION
Gentamicin is an antibiotic belongs to the family of
aminoglycosides that was discovered in 1963 and was isolated
from Micromonospora purpurea. It is readily soluble in water
due to the presence of hydroxyl groups on its chemical structure,
but practically insoluble in organic solvents (Šoltés, 1999).
The narrow therapeutic range possesses by gentamicin has made
* Corresponding Author
Farahidah Mohamed, Kulliyyah of Pharmacy (KOP), International
Islamic University Malaysia (IIUM), Jalan Sultan Ahmad Shah, Bandar
Indera Mahkota, 25200 Kuantan, Pahang Darul Makmur, Malaysia.
Tel: +6095716400 Fax: +6095716775, Email: farahidah@iium.edu.my
the antibiotic to be one of the heated debate topics amongst the
physicians, whether it should not be used at all or merely that it
should not be used extensively (Ariano et al., 2008). Ototoxicity
and nephrotoxicity have been reported widely to be the main
concern whenever the aminoglycosides are administered to the
patients (Šoltés, 1999; Ariano et al., 2008; Al-Hamad, 2014; Yusof
et al., 2014). This concern has become the driving factor for many
researchers to develop a method that can minimize the
bioavailability of aminoglycoside systemically but at the same time
maximize the therapeutic effect only where it is needed.
A carrier system with sustained release characteristic is the
common option available to overcome the problem (Chung and
Huang, 2001; Balmayor et al., 2012; Abed and Couvreur, 2014).

© 2016 Ahmad Fahmi Harun Ismail et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-
ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).
08 Ismail et al. / Journal of Applied Pharmaceutical Science 6 (01); 2016: 007-014

There is a quite considerable amount of literature has Supplies, England) was used. All solvents were purchased from
been published pertaining the sustained release of gentamicin Merck (Germany) with analytical grade. Staphylococcus aureus
using biodegradable polymers such as poly(lactic-co-glycolic acid) (ATCC 29523) was purchased from Microbiologics® (USA).
or commonly known as PLGA (Friess and Schlapp, 2002; Schlapp
and Friess, 2003; Ismail et al., 2012). The quantification method Instrumentation
was developed and validated based on the requirements listed by The analysis was done using single beam UV-
The International Conference on Harmonization of Technical spectrophotometry module (U-1900 Toshiba, Japan) with
Requirements for Registration of Pharmaceuticals for Human Use wavelength scanned from 600 nm to 190 nm. The silica quartz
(ICH). Although gentamicin has been extensively used as the square cell cuvette (Optima, Japan) with transmittance of 51.4 ±
subject for sustained release formulations, literature reviews have 0.3% at wavelength 545 nm was used throughout the whole
indicated that there is no general agreement about developing an experiment.
analytical method validation (AMV) for gentamicin by using UV-
spectrophotometer. It is a rapid and cost effective with EXPERIMENTAL METHODS
significantly moderate sensitivity in detecting gentamicin using
Colorimetric Reagent
UV-spectrophotometry as compared to analytical methods using
Accurately 50 mg of ninhydrin powder was weighted and
HPLC or GC-MS.
dissolved with 10 mL of phosphate buffered saline (PBS) solution
When it comes to the relation between transmittance and
(pH 7.4) to get the stock solution with the concentration of 5
absorbance of a solution in the application of UV-
mg/mL. The stock solution was kept under 4 oC and was protected
spectrophotometry detection, Beer-Lambert Law is one of the
from any light source.
basic principles in understanding the concept. Briefly, the strength
of one light beam passes through a translucent cell or cuvette
Fabrication of Gentamicin Microparticles
holding an absorbing solution will have an inversely proportion
Modified double emulsion solvent evaporation method
between the intensity of the beam detected and the concentration
was adopted from Ismail et al. (2012). Briefly, PLGA was
of the solution (Behera et al., 2012; Ismail et al., 2015). It was
dissolved in 2 mL of chloroform before mixed up with 200 µL
reported elsewhere that the concentration of gentamicin was
aqueous solution containing 100 mg of gentamicin to be regarded
determined by using fluorescent assay at the wavelength of 450
as the primary emulsion. Secondary emulsion was achieved by
nm, with the R2 value for the linearity constructed at this
mixing between primary emulsion 8 mL of 1% w/v PVA solution,
wavelength was less than 0.98 (Lecároz et al., 2006). Additionally,
before it was transferred into the hardening tank (100 mL of 1%
the same literature is also reported that the correlation for linearity
w/v PVA solution) for solvent evaporation process which took 3
constructed using HPLC for gentamicin extracted from
hours. The mixing process for primary and secondary emulsions
microparticles using dichloromethane and sodium hydroxide gives
was done by using ultra sonicator (QSonica, USA; 3 cycles for 3
the values of 0.889 and 0.855 respectively.
seconds each at 50 MHz). The microparticles were collected by
The aim of this study is to develop a method to quantify
mean of centrifugation (4000 rpm, 5 min) and followed by
the presence of gentamicin in the form of gentamicin-ninhydrin
washing to remove PVA before they were lyophilized under
complex by using UV-spectrometer. The method would be
reduced pressure.
developed and validated based on the requirements listed under
ICH Q2 (R1) guidelines. In addition, the study was done to prove Table 1: The nine formulations of the microparticles based on the ratio and the
the stability and the efficacy of the gentamicin for post fabrication blends of PLGA used. The amount of gentamicin loaded for each formulation
process by evaluating the response of the antibiotic against was 100 mg each.
staphylococcus aureus. Formulation Intrinsic Viscosity (dL/g) PLGA ratio
F1 0.2/0.4 50:50
It is believed that the analytical method developed and F2 0.2/1.0 50:50
validated in this study would provide a rapid and cost effective in F3 0.4/1.0 50:50
vitro quantification for gentamicin. F4 0.2/0.4 75:25
F5 0.2/0.4 25:75
F6 0.2/1.0 75:25
MATERIALS F7 0.2/1.0 25:75
F8 0.4/1.0 75:25
Chemicals and Reagent F9 0.4/1.0 25:75
Gentamicin sulphate was purchased from local
pharmacy. Purasorb® PLGA with three different intrinsic The gentamicin loaded microparticles were evaluated
viscosities; 0.2 dL/g (14 kDa), 0.4 dL/g (34 kDa) and 1.0 dL/g based on the particle size distribution and the amount of drug
(100 kDa) were purchased from Purac (Netherland). Ninhydrin entrapment. For this study, nine different formulations were
with laboratory reagent grade was purchased from Fisher selected based on the combinations of the PLGA molecular
Scientific (United Kingdom). Poly Vinyl Alcohol (PVA) with weights and the ratio used between those molecular weight blends
molecular weight approximately 115 kDa (BDH Laboratory (Table 1).
 Ismail et al. / Journal of Applied Pharmaceutical Science 6 (01); 2016: 007-014
Particle Size Distribution
The particle size distribution was evaluated by using
Laser Particle Size Analyzer (Bettersize Instruments, China) and
was done in triplicate. The particles were suspended in deionised
water before subjected for measurement.
Encapsulation Efficiency
Briefly, 5-8 mg of gentamicin loaded microparticles were
placed in a tube and re-dissolved using 1 mL of chloroform
followed with the addition of another 1 mL of PBS solution (pH
7.4).
This method was adopted from Ismail et al. (2012) and
modified accordingly. The tube was rotated end-to-end for 1 hour
before 800 µL of the aqueous part was extracted and transferred
into another tube after centrifugation process (4000 rpm, 5 min).
Equal amount of colorimetric reagent was added to the solution
before it was subjected to heat treatment using water bath (95 oC,
15 min) and followed with cold treatment using ice cold water
bath for 10 minutes. This final solution was subjected to UV-
spectrophotometry evaluation with the wavelength of 418 nm. The
quantification for each formulation was done in triplicate and the
average values were recorded.
Gentamicin Efficacy for Post Microparticle Fabrication
In short, 10 mg of gentamicin loaded microparticles were
dissolved in 1 mL chloroform and another 1 mL of PBS solution
was added. End-to-end rotation was done for 1 hour to ensure all
the microparticles were dissolved before the aqueous part was
extracted. 100 µL from the extracted solution was dropped on a
blank paper disk and tested against staphylococcus aureus. The
disk diffusion method adopted and modified from Ismail et al.
(2012) was utilized by preparing the lawn of S. aureus by using
nutrient agar. Optical density of the bacteria broth was measured
using UV-spectrometer (Hitachi U1900 Single Beam, Tokyo
Japan) which corresponded to 0.5 McFarland standard. The
concentration of 108 colony forming unit (CFU)/mL (0.5
McFarland Standard) was achieved by measuring the optical
density which was between 0.008-0.1 absorbance value (Andrews,
2001).
The S. aureus was cultured by using nutrient broth and
the nutrient agar was used as the medium for the bacteria lawn.
The zone of inhibition for each formulation of microparticles was
measured by using Absolute Digimatic Caliper Mitutoyo (Japan)
with sensitivity up to 0.01 mm.
METHOD DEVELOPMENT AND VALIDATION
Preparation of Gentamicin Stock Solution
Accurately 1 g of gentamicin powder was weighted and
dissolved with 10 mL of PBS solution (pH of 7.4) and stored at 4
o same wavelength used to quantify the samples. There are specific
C. This stock solution with final concentration of 100 mg/ml
(10% w/v) was diluted accordingly whenever required. The PVA
1% w/v was prepared by dissolving the PVA powder by using
ultra pure water.
09
Specificity
Gentamicin-ninhydrin complex in PBS would give a
purple colour with clear non turbid solution. Gentamicin alone in
aqueous solution would not give any colour at relatively low
concentration and could not be detected with UV
spectrophotometer. Initially, a wavelength scan was performed to
get the best peak for gentamicin-ninhydrin complex without any
possible interference with other possible components in the
solution. PBS solution was used as blank solution and was
regarded as background reading. The same scanning process was
done for PLGA and ninhydrin alone to eliminate any false positive
in selecting the best peak for gentamicin-ninhydrin complex. The
determined wavelength would be used throughout the study
whenever needed.
Linearity and Standard Curve
The linearity of the six different known concentrations of
gentamicin-ninhydrin complex was subjected to the previously
determined wavelength. The absorbance of the respected
concentrations was recorded. A concentration vs. absorbance
graph was plotted on which the equation 𝑦 = 𝑚𝑥 + 𝑐 and the R2
were established. The concentration of the analyte was determined
with the notation of 𝑥 with 𝑦 is the absorbance value. The
correlation coefficient R2 value for all the standard curves
constructed for this study was set to be not less than 0.995 as the
one of the acceptance criteria (Rajesh et al., 2014; Ismail et al.,
2015).
Accuracy and Precision
For the accuracy, four known different concentrations of
gentamicin-ninhydrin complex were subjected to the established
wavelength (418 nm) and the experimental concentrations were
recorded. Each concentration was subjected to the wavelength in
triplicate and the mean and standard deviation were recorded. The
precision was evaluated by taking into consideration the
percentage deviation of the absorbance values obtained from the
actual concentrations. The interday precision was carried out by
doing the measurement in 3 different days for 3 different
preparations. The intraday precision on the other hand was done
by subjecting four known sample concentrations to the same
wavelength (418 nm) but in 3 different specific time points on the
same day. The percentage recovery should not be less than 98%
and more than 102% with % RSD value is smaller than 2.0% to
meet the acceptance criteria (Rajitha et al., 2011; Ismail et al.,
2015).
Limit of Detection (LOD) and Limit of Quantification (LOQ)
The evaluation for both LOD and LOQ was done by
subjecting the blank solution (PBS solution with pH of 7.4) to the
calculations to determine both LOD and LOQ as described below;
3.3 𝑥 𝑆.𝐷 10 𝑥 𝑆.𝐷
LOD = LOQ =
𝑚 𝑚
010 Ismail et al. / Journal of Applied Pharmaceutical Science 6 (01); 2016: 007-014

S.D or standard deviation in the formula was referring to the
standard deviation of the absorbance values of the blank and 𝑚 is
the slope of the standard curve constructed previously (Iqbal et al.,
2013). All readings for LOD and LOQ were taken 3 times.
Robustness
The robustness of the method was determined by
intentionally introducing small changes in the procedures or
sample preparations. The deviations of data collected for
robustness were compared to the original data and any significant
changes were recorded. The wavelength was intentionally changed
to 421 nm, 415 nm and 400 nm and the standard curves for each
wavelength were constructed with the correlation coefficient R2
values were recorded. In addition, the second analyst was assigned
to construct the standard curve to get the R 2 values and the linear
equations based on the absorbance values of six different known
microparticles. It was obvious that higher molecular weight
(higher intrinsic viscosity) with higher ratio would trap more
gentamicin as compared to the lower molecular weight of PLGA.
Although the contradiction was observed, this finding
was recorded with a fabrication system employed the single
polymer emulsion and mechanical shearing force. The current
study however had utilized the ultrasonic wave as the mean of
homogenizing the emulsion in addition to the employment of
molecular weight blends of the polymer used. The emulsion is
more stable when the polymer used is high in molecular weight.
Thicker emulsion will be formed with high molecular weight
polymer as compared to low molecular weight polymer. Hence,
the droplets formed are more stable with less coalescence rate
between the droplets.
50 42.98

Encapsulation Efficiency
concentrations of gentamicin-ninhydrin complex. The method was 45 41.42
32.85 38.03 33.87
considered to be robust if the R2 values obtained were not less than 40 30.62 32.82 35.33
0.995. 35
30

(%)
25
RESULT AND DISCUSSION 20 11.64
Particle Size Distribution and Encapsulation Efficiency 15
10
The size distribution for all formulations indicate that the
5
size of the particles fell in the range between 1.624 µm ± 0.194 to 0
5.103 µm ± 1.537 (Table 2). Relatively, the size distribution of the
F1 F2 F3 F4 F5 F6 F7 F8 F9
microparticles for all formulations could be considered as
homogenously dispersed. This homogenicity was determined Formulation
Fig. 1: Encapsulation efficiency of the gentamicin loaded microparticles based
based on the span values for each of the formulations. The span
on the formulations employed.
value was calculated based on the size distributions of the particles
at 3 different distributions (10%, 50% and 90%) with the Table 2: The size distribution of the gentamicin loaded microparticles
formulation as described as follow: according to the formulations.
𝐷90 − 𝐷10 Size Distribution (µm) Span
Formulation S.D (± µm)
𝑆𝑝𝑎𝑛 𝑣𝑎𝑙𝑢𝑒 = (n=3) Value
𝐷50 F1 4.973 0.427 0.282
Whereby D90, D10 and D50 are the particle distribution F2 2.700 0.394 0.299
at 90%, 10% and 50% respectively. The span value lower than 1 F3 2.346 0.246 0.400
F4 5.103 1.537 0.239
indicates that the size distribution can be considered as F5 2.973 0.127 0.353
homogenously dispersed. Although the particle size was F6 2.932 0.432 0.292
homogenously distributed, the size distribution was not really a top F7 1.624 0.194 0.426
F8 1.785 0.369 0.430
priority in this study since the microparticles were intended to be F9 3.244 0.792 0.305
used locally at the site of infection without having to go through
any parenteral routes. The results of the encapsulated efficiency Gentamicin Efficacy for Post Microparticle Fabrication
(EE) indicate that the molecular weight and the PLGA blends The fabrication process to encapsulate gentamicin proved
influenced the amount of gentamicin encapsulated using this to be suitable and compatible without changing the efficacy of
fabrication method. Double emulsion solvent evaporation method drug itself. The efficacy for post fabrication process was evaluated
is meant to encapsulate drugs which are hydrophilic in nature such by extracting the encapsulated gentamicin and subjecting to the
as gentamicin (Ismail et al., 2012). Figure 1 showed that although bacteria lawn. Figure 2 showing the antimicrobial action of
gentamicin was able to be encapsulated within the microparticles, gentamicin against the S. aureus. It was found that the efficacy of
the highest amount of gentamicin encapsulated was 42.98% ± 2.60 gentamicin was still intact based on the formation of zones of
for F8. The lowest EE (11.64% ± 2.66) however represented by inhibition. Although the diameter of the zones of inhibition could
the formulation (F1) employed 0.2 dL/g and 0.4 dL/g of PLGA be measured according to the formulations used, it was not
with the ratio of 50:50. Among the plausible explanations for this scientifically suitable to represent the data to correspond the
finding is that the molecular weight of the PLGA plays a vital role amount of drug loading unless all the variables were strictly
in determining the amount of gentamicin encapsulated in the controlled. Interestingly based on the result obtained, it was
 Ismail et al. / Journal of Applied Pharmaceutical Science 6 (01); 2016: 007-014 011

proven that the encapsulated gentamicin could still be effective fabrication was characterized by using UV-spectrophotometer.
against the S. aureus. The diameters of the zone of inhibition Since gentamicin is poorly absorbed by the visible light and UV-
according to the formulation were tabulated under Table 3. light, an indirect spectrophotometry method needed to be
Gentamicin has the ability to bind to a conserved sequence of developed.
rRNA of the bacteria which is very close to the site of codon- Ninhydrin was used to produce a colorimetric procedure
anticodon recognition of the 30S subunits (Yoshizawa et al., in order to quantify the gentamicin encapsulated. The principle
1998). reaction between ninhydrin and gentamicin is based on the
This binding would reduce the accuracy of the bacteria in chemical interaction of ninhydrin with the primary and secondary
translating the genetic material and inhibit the translocation of the amine groups present in the gentamicin chemical structure which
ribosomes which interrupting the synthesis of protein materials. produces purple colour (Frutos et al., 2000). Spectrophotometric
This polycationic inhibitor of the 30S ribosomal subunit would method with rapid, sensitive and cost effective for analysis of a
cause the bactericidal against S. aureus by disrupting the cell single component in solution can be achieved for any drugs that
membrane transportation and the permeability of the cell wall obey the Beer-Lambert Law.
(McLawhon, 2012). Based on the wavelength scanning done for the
gentamicin-ninhydrin complex, the wavelength of 418 nm was
Table 3: The average diameters of zone of inhibition according to the
formulations. selected due to the colorimetric appearance of the solution (clear
Average Diameter Standard Deviation purple) which indicated the detection should not be under UV
Formulation
(mm) (±mm) region (below 400 nm). In addition, the scanning spectrum from
F1 24.78 1.36
F2 23.22 2.88
600 nm to 200 nm verified that at 418 nm region, the detection
F3 22.37 0.39 would not be interrupted with the wavelength of PLGA and
F4 25.60 1.80 ninhydrin alone itself (Figure 3).
F5 25.02 1.88
F6 25.82 0.91 Although the 418 nm wavelength was determined by the
F7 25.03 0.62 UV module, the gentamicin-ninhydrin complex was also subjected
F8 22.30 1.68 to 400 nm to evaluate the degree of deviation by constructing the
F9 24.20 2.75
linearity and obtaining the correlation coefficient R 2 value. Based
on the R2 value obtained from the constructed linearity at 400 nm,
it was below the acceptance requirement (0.9772) which is not less
than 0.995. This undesired correlation coefficient value had made
the wavelength at 418 nm as the best choice for the specificity in
detecting gentamicin-ninhydrin complex.

Linearity and Standard Curve

Fig. 2: The zone of inhibition for some of the formulations (F2, F6 and F7)
gentamicin against Staphylococcus aureus.
Specificity
In this study, gentamicin loaded microparticles were
fabricated using PLGA and the amount of drug loading for post
.
The linearity was determined based on the constant direct
proportionate relation between the concentration of gentamicin-
ninhydrin complex and the absorbance values specifically at 418
nm wavelength.
Six known concentrations of gentamicin-ninhydrin
complex were prepared (2.0, 4.0, 5.0, 6.0, 8.0 and 10.0 mg/mL)
and subjected to 418 nm to get the absorbance values. The
standard curve obtained from the UV-spectrophotometry based on
these 6 concentrations was in linear form with the equation and the
R2 of 𝑦 = 0.069𝑥 + 0.007 and 0.9998 respectively. The standard
curve is illustrated in Figure 4.
The readings for each concentration were done in
triplicate and the average of those three was calculated to
maximize data reliability. The inconsistency of the readings from
the UV module could be neglected since the background solvent
used in this study was PBS solution. It was mentioned by Ismail
and co-workers that volatile organic solvent such as
dichloromethane used as the background solution would contribute
to the inconsistency of the readings obtained due to the rapid
evaporation of the solvent itself (Ismail et al., 2015).
012 Ismail et al. / Journal of Applied Pharmaceutical Science 6 (01); 2016: 007-014
Fig. 3: The wavelength scanning spectrum of PLGA, ninhydrin (NH) reagent, and gentamicin-ninhydrin complex for specificity.
0.8
0.7
0.6
Absorbance
0.5
0.4
0.3
0.2
0.1
0 the module for such over concentrated sample would drop below
0 2 4 6 8 10
Gentamicin-ninhydrin Concentration (mg)
Fig. 4: The standard curve constructed for linearity study.
Accuracy and Precision
The intermediate precision was carried out by using 4
known concentrations of the gentamicin-ninhydrin complex (2.0,
4.0, 6.0 and 8.0 mg/mL) and was measured in 3 consecutive days.
The 4 known concentrations were freshly prepared each day and
for each concentrations, the readings were taken in triplicate.
Based on the data for intermediate precision (Table 4), the
quantification method was considered to be precise in detecting
gentamicin-ninhydrin complex at different concentrations as the
%RSD obtained was less than 2.0%. This similar finding was
observed and reported by a few others with the same acceptance
criteria monitored (Behera et al., 2012; Ismail et al., 2015).
For repeatability study (intraday precision), similar
outcome was observed where 3 subsequence readings were taken
on the same day. All 4 known concentrations (2.0, 4.0, 6.0 and 8.0
mg/mL) were freshly prepared each time. The repeatability data
was tabulated in Table 5 with the %RSD for all concentrations
shown the values less than 2.0%. This quantification method was
.
considered to be precise and met the requirement set by ICH Q2
(R1) guidelines. The accuracy study for this method was modified
due to the high intensity of purple color if the concentration of
gentamicin-ninhydrin complex was raised up to 100% w/v and
more.
The absorbance reading would jump over 2.0 when the
transparency of the solution dropped drastically. The detection of
12 0.1% for the transmittance.
Low transmittance resulted from too concentrated sample
would give a considerably high standard error in the reading.
Consequently, the same concentrations were used (2.0, 4.0, 6.0
and 8.0 mg/mL) in determining the accuracy of the method
without compromising the requirement of acceptance of the data
collected.
Almost the same issue was described in another study
done elsewhere with the quantification method developed for
Nigella sativa oil using UV spectrophotometry (Ismail et al.,
2015). Table 6 indicates the accuracy data collected with each
concentration was done in triplicate. The average recovery was in
between the acceptance range (98% to 102%) with the %RSD was
less than 2.0%.
Limit of Detection (LOD) and Limit of Quantification (LOQ)
The LOD and LOQ were calculated based on the
formulations mentioned under the experimental methods. By
considering the control experimental condition for this study, the
LOD and LOQ values were 0.016 mg/mL ± 0.0003 mg/mL and
0.196 mg/mL ± 0.001 mg/mL respectively with the slope of the
standard curve was 0.07. The correlation coefficient R 2 for the
standard curve was 0.9998.
 Ismail et al. / Journal of Applied Pharmaceutical Science 6 (01); 2016: 007-014 013

Table 4: The intermediate precision was obtained by using UV-spectrophotometry for gentamicin-ninhydrin complex.
Concentration (mg/mL) Average Actual Concentration (mg/mL) (n=3) S.D R2 For Standard Curve Precision (%RSD)
Day 1 0.9998
2.000 2.031 0.003 0.159
4.000 4.103 0.003 0.068
6.000 6.054 0.007 0.108
8.000 8.058 0.015 0.183
Day 2 0.9995
2.000 2.039 0.001 0.035
4.000 4.055 0.001 0.008
6.000 6.055 0.001 0.007
8.000 8.054 0.001 0.012
Day 3 0.9996
2.000 2.073 0.006 0.266
4.000 4.139 0.027 0.661
6.000 6.202 0.068 1.098
8.000 8.254 0.031 0.374

Table 5: The repeatability study (intraday precision) by using UV-spectrophotometry.

Concentration (mg/mL) Average Actual Concentration (mg/mL) (n=3) S.D R2 For Standard Curve Precision (%RSD)
1st Reading 0.9998
2.000 2.036 0.007 0.332
4.000 4.020 0.010 0.239
6.000 6.040 0.009 0.147
8.000 8.130 0.002 0.027
2nd Reading 0.9998
2.000 2.043 0.002 0.114
4.000 4.043 0.004 0.097
6.000 6.056 0.017 0.285
8.000 8.062 0.008 0.102
3rd Reading 0.9998
2.000 2.031 0.003 0.159
4.000 4.103 0.003 0.068
6.000 6.054 0.007 0.108
8.000 8.058 0.015 0.183

Table 6: The accuracy of the method was evaluated based on the average recovery and the %RSD value of each concentration.
Nominal Concentration (mg/mL) Actual Concentration (mg/mL) (n=3) Average % Recovery S.D %RSD
2.000 2.009 100.448 0.847 0.844
4.000 4.021 100.516 1.118 1.113
6.000 6.094 101.561 1.376 1.355
8.000 8.056 100.701 0.120 0.119

Table 7: The correlation coefficient R2 values and the linear equations.

Condition Linear Equation R2 Value
421 nm Wavelength 𝑦 = 0.070𝑥 + 0.005 0.9991
415 nm Wavelength 𝑦 = 0.069 + 0.009 0.9993
Second Analyst 𝑦 = 0.069 + 0.004 0.9996

Robustness quantify gentamicin-ninhydrin complex was robust enough to

The wavelength determined by the UV module was
modified with small adjustment and the same concentrations used
in constructing the linearity were subjected to the new wavelength.
For robustness, the actual wavelength was adjusted to 421 nm and
415 nm before the linearity for both wavelengths was determined.
From the data in Table 7, it is apparent that small changes done
intentionally to the developed method is not affecting the
reliability in quantifying the concentration of gentamicin in the
given solution. Although the changes were done with the values of
+3 nm and -3 nm from the actual wavelength, R2 values obtained
for both adjusted wavelengths were not less than 0.995. Similar
observation was acquired with the construction of standard curve
by second analyst. The R2 value was 0.9996 and still above
acceptance limit. Hence, this proved that the method developed to
stand small intentional changes.
CONCLUSION
The quantification method for gentamicin in the form of
gentamicin-ninhydrin complex was developed and validated by
using UV-spectrophotometry. The basic requirements for the
acceptance criteria underlined by ICH Q2 (R1) guidelines where
met and fulfilled. The exploration of the relationship between the
concentration of gentamicin and the UV absorbance value in order
to develop an analytical method for in vitro rapid quantification
was successful. Therefore in summary, the current study suggested
that this analytical method was rapid, precise, specific, accurate,
robust and cost effective to be used in quantifying gentamicin for
in-vitro assessment. In addition, the microparticles fabrication
014 Ismail et al. / Journal of Applied Pharmaceutical Science 6 (01); 2016: 007-014
process was proven to be suitable in encapsulating gentamicin by
using PLGA without compromising the efficacy of the antibiotic
itself.
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How to cite this article:
Ismail AFH, Mohamed F, Rosli LMM, Shafri MAM, Haris MS,
Adina AB. Spectrophotometric Determination of Gentamicin
Loaded PLGA Microparticles and Method Validation via
Ninhydrin-Gentamicin Complex as a Rapid Quantification
Approach. J App Pharm Sci, 2016; 6 (01): 007-014.